In vitro evaluation of three engineered multispecies endodontic biofilms on a dentinal disk substrate

The aim of this study was the development of a complex multispecies endodontic biofilm using Candida albicans, Proteus mirabilis and Pseudomonas aeruginosa on a biofilm of Enterococcus faecalis in a dentinal substrate design. The endodontic pathology is a biofilm-mediated infection, and the aim of root canal therapy is to reduce, as much as possible, the bacterial population. Thus, it is important to develop a laboratory endodontic biofilm to test the effect of new irrigation and obturation techniques on reduction of bacterial count. The culture of Enterococcus faecalis from ATCC 29212 began with aerobic cultivation on blood agar, followed by transfer to Brain Heart Infusion (BHI) broth with 5% sucrose. Incubation occurred in a shaker at 37 °C for 24 h, followed by an additional 24-h static phase. After 10 d, Proteus mirabilis, Pseudomonas aeruginosa, and Candida albicans were introduced sequentially in three distinct groups. Group 1: the order of addition was Candida albicans, Proteus mirabilis, and Pseudomonas aeruginosa; Group 2: the order was Pseudomonas aeruginosa, Candida albicans, and Proteus mirabilis; and Group 3: Proteus mirabilis, Pseudomonas aeruginosa, and Candida albicans. After 16 days, the biofilm was carefully extracted, transferred to sterile BHI, and dissected using a sterile needle technique. Subsequently, an optical density test, bacterial counts, and colony enumeration were performed on various agar plates. Group 2 in which Pseudomonas aeruginosa was added directly after Enterococcus faecalis followed by Candida albicans and Proteus mirabilis showed significantly greater total bacterial count than the other two groups.